Gliogenesis and maintenance of glial cell types are critical to development and function of the nervous system. Sox10 is a transcription factor that is essential for development of multiple glial lineages including oligodendrocytes in the central nervous system as well as neural crest- derived Schwann cells, satellite glia and enteric nervous system neurons and glia in the periphery. Regulatory regions from the Sox10 gene have previously been used to drive expression of single fluorophore transgenes for tracking the migration of glial progenitors and the distribution of mature glial cell types. These first generaton tools have been valuable but have not permitted concurrent imaging of cell nucleus, morphology, or signaling between individual cells. Calcium signaling is a fundamental cellular mechanism by which cells transmit intracellular signals in response to extrinsic stimuli or transmit signals to adjacent cells and is essential for many aspects of glial cell development and maintenance. Studies of these processes have been primarily investigated in cell culture systems that are amenable to loading with fluorescent dyes or transfection by exogenous plasmids. However recent progress in development of Genetically Encoded Calcium Indicators (GECIs) has produced fluorescent reporters that allow monitoring of calcium transients in living cells and organisms. In the context of the R21 mechanism we propose generation of a multi-cistronic transgenic allele of Sox10 in mice as a novel tool for imaging migration, cell morphology and signaling between glial cells. In Specific Aim 1 we will construct and test multi-cistronic expression vectors in vitro to identify the optimal combination of reporters to monitor calcium signaling, nuclear localization and cell morphology. In Specific Aim 2 we will incorporate a multi-spectral expression cassette into a Sox10 bacterial artificial chromosome backbone and establish transgenic lines that recapitulate endogenous Sox10 expression in vivo. The ability to concurrently track migration, cell morphology and calcium signaling among glial populations will significantly impact the field by enabling analysis of developmental mechanisms that are relevant for directed differentiation of progenitors cells and will enable pharmacologic analyses to identify potential therapeutic agents for treatment of central and peripheral neuropathies.